FIGURE 2:
Ubp2 and Ubp15 regulate the degradation of ARTs. (A) art4Δ and art4Δ ubp2Δ ubp15Δ yeast cells carrying Art4-FLAG, Art4-UL36-FLAG, or Art4-UL36C40S-FLAG expression vector. Protein extracts were analyzed by SDS–PAGE and immunoblot. (B) Immunoblot analysis of chromosomal Art4-FLAG in WT and ubp2Δ ubp15Δ yeast cells carrying functional or catalytically dead Ubp2/Ubp15 expression vector. (C) WT and ubp2Δ ubp15Δ yeast cells expressing chromosomal Art4-FLAG or ubp2Δ ubp15Δ cells overexpressing (OE) Art4-FLAG (driven by the CYC1 promoter inserted into ART4 locus) were treated with 100 μg/ml CHX to stop protein synthesis. Protein extracts from the indicated time points were analyzed by SDS–PAGE and immunoblot. (D) Art4 levels (relative to t = 0) as in C were quantified by the LI-COR system. Data from three independent experiments are presented as mean ± SD. (E, F) similar to C and D, except that yeast strains with chromosomal Art1-FLAG were examined.